Anaplasma phagocytophilum, is a human pathogen that infects neutrophils and causes Human Granulocytic Ehrlichiosis. In the Unites States it is the second most common tick-associated infection, only surpassed by Lyme disease. Although neutrophils are very efficient in killing bacteria, A. phagocytophilum is the exception as it not only escapes killing by neutrophils, it lives and replicates inside neutrophils. A. phagocytophilum interferes with superoxide-mediated killing and delays neutrophil apoptosis. It is poorly understood how A. phagocytophilum accomplishes these major feats. This proposal seeks to identify the basic mechanism how A. phagocytophilum manipulates these cellular processes. Our recent identification of the first bacterial virulence factor in A. phagocytophilum, AnkA, may play a pivotal role in manipulating neutrophil functions. Consequently, A. phagocytophilum infection of neutrophils provides an ideal model to study the pathways involved in neutrophil biology and bacterial killing. The global hypothesis is that translocated AnkA interferes with host signaling pathways in the neutrophil, resulting in the inhibition of superoxide generation and the delay of apoptosis. Upon bacterial binding to the host cell, AnkA is translocated into the host cell cytoplasm and is then rapidly tyrosine phosphorylated (within seconds to minutes). Tyrosine phosphorylation allows AnkA to bind to SH2 domains of key signaling proteins in the host cell. Further, AnkA may also bind proteins via SH3 domains and ankyrin repeats. Through these putative protein interactions AnkA may alter host cell signaling and cause the cellular changes in neutrophils. Aim 1 seeks to identify the specific interactions of host cell proteins that bind to AnkA Aim 2 proposes to study the role of AnkA in inhibition of killing by the neutrophil. Aim 3 will examine the role of AnkA in the delay of neutrophil apoptosis. The long-term goals are: 1) the identification of the mechanism leading to survival of A. phagocytophilum in neutrophils, 2) the development new avenues to manipulate neutrophils, leading to potential new treatments of inflammation or infections, and 3) enhancement of our understanding of neutrophil biology as it relates to the immune system. PUBLIC HEALTH RELEVANCE: Human Granulocytic Ehrlichiosis, caused by A. phagocytophilum, is the second most common tick- associated disease in the United States. This proposal seeks to investigate how A. phagocytophilum can survive in neutrophils. This may lead to a better understanding of the function of neutrophils within the immune system and may lead to potential new treatments of inflammation and infections.